THE SYSTEM OF MONITORING OF INHIBITORY DRUG RESIDUES IN RAW COWS' MILK IN SLOVAKIA
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Potravinarstvo Slovak Journal of Food Sciences
Potravinarstvo Slovak Journal of Food Sciences
vol. 15, 2021, p. 858-868
https://doi.org/10.5219/1687
Received: 1 September 2021. Accepted: 25 September 2021.
Available online: 28 October 2021 at www.potravinarstvo.com
© 2021 Potravinarstvo Slovak Journal of Food Sciences, License: CC BY 4.0
ISSN 1337-0960 (online)
THE SYSTEM OF MONITORING OF INHIBITORY DRUG RESIDUES
IN RAW COWS' MILK IN SLOVAKIA
Peter Zajác, Stanislava Zubrická, Jozef Čapla, Jozef Čurlej, Maroš Drončovský
ABSTRACT
This article provides brief information on the system of monitoring raw cows’ milk for the presence of inhibitory veterinary
drug residues in the Slovak Republic. We are describing in detail how the monitoring is carried out and what laboratory
methods are used for this monitoring. We also deal with the issue of the disposal of contaminated milk. The presence of
inhibitory veterinary drugs like antibiotics or residues of these drugs in milk in an amount exceeding maximal residual limits
defined in legislation is illegal. Milk supplies containing detectable concentrations are not acceptable. The reputation of milk
as a healthy and safe food should be protected. Dairy companies and consequently consumers want to be confident that milk
and milk products are free of inhibitory veterinary drugs contamination. Small amounts of certain antimicrobial agents may
affect antibiotic resistance in the human population, some percentage of the population is hypersensitive to antibiotics and
other drugs. The presence of drug residues in milk affects the technological process of the production of fermented dairy
products. Each country should implement a system of monitoring these drugs' residues in food. The system, which is applied
in Slovakia, is effective, it can detect and the presence of these substances before milk processing in dairies. The total
incidence of these substances in 2020 was 0.025% of all 12,181 samples tested in central testing laboratories. During the last
20 years.
Keywords: raw milk; drug residues; cow milk; antibiotics
INTRODUCTION In the Slovak Republic, therefore, food business operators
The system of monitoring of raw cow's milk for the ensure compliance with the requirements of this Regulation
presence of residues of inhibitory substances in Slovakia is through a multi-level control system.
performed in several stages of the food chain. This is a The use of antibiotic therapy to treat and prevent udder
mandatory obligation arising from Commission Regulation infections in cows is a key component of mastitis control in
(EC) No 1662/2006 (EC, 2006). For this Regulation, food Slovak Republic (Zajác, Golian, Vácziová, 2006; Zajác,
business operators must initiate procedures to ensure that Golian, Čapla, 2007a; Zajác, Golian Vácziová, 2007b;
raw milk is not placed on the market if: Kološta, 2007), Czech Republic (Navrátilová, 2008) and
a) it contains antibiotic residues in quantities which, many other countries (Hillerton et al., 1999).
in the case of any of the substances listed in Factors to be considered to choose the most suitable
Annexes I and III to Regulation (EEC) No. method of residue detection are the type of antibiotic used,
2377/90 (EEC, 1990) exceeds the value permitted expected time limitations, sensitivity of the test, and costs
under that Regulation, or (Senyk et al., 1990).
b) the combined total antibiotic residue level exceeds The antibiotic residue detection assay systems that are
any maximum permitted level. currently available use different methods and test organisms
Regulation EEC No. 2377/90 (EEC, 1990) was repealed (Van Eenennaam et al., 1993).
by regulation (EC) No. 470/2009 (EC, 2009a), but the Microbiological assays for the detection of antibiotic
anexes present in repeald regulation remain in force. residues utilize the genus Bacillus sp. because of its high
If raw milk does not meet these conditions, the food sensitivity to the majority of antibiotics. The Bacillus
business operator must, following Commission Regulation stearothermophilus var. calidolactis is routinely used by the
(EC) No. 1662/2006 (EC, 2006) to notify the appropriate dairy industry to screen antibiotic residues. Delvotest SP is
authority (Regional Veterinary and Food Administration of a multiple microbial inhibitor test used to detect
the Slovak Republic) and take measures to immediately antimicrobial agents such as beta-lactams and sulpha
solve this problem. compounds (Suhren and Beukers, 1998).
Volume 15 858 2021Potravinarstvo Slovak Journal of Food Sciences
Microbiological tests Copan Milk Test and Delvotest SP- - CMT+ or formally Copan Milk test (Supplier:
NT can be used for screening antimicrobial substances in Climax, s.r.o., Bratislava);
milk (Le Breton, Savoy-Perroud and Diserens, 2007). - Twinsenzor (Supplier: Merck Slovakia);
In general monitoring of antimicrobial agent residues, To determine residues of inhibitory substances, the
microbial growth inhibition methods, and rapid tests are instructions provided by the suppliers and requirements of
used. Microbial inhibitor screening methods are easy to the ISO/TS 26844 (ISO, 2006) were fulfilled.
perform and enable the detection of a wide spectrum of In the Delvotest® SP-NT ampoules (Figure 1) with a
agents. The microbial inhibitor screening methods do not culture medium containing Bacillus stearothermophilus
attain with some antibiotics the sensitivity at the levels var. calidolactis were used. The ampoules were incubated
specified by the MRLs (maximum residue level). For the for 3 h at 64 ±0.5 °C and the color change was evaluated.
determination of these agents, other methods should be used Samples were considered as negative if the agar change the
(e.g. immunochemical or receptor). Another disadvantage color from violet to yellow (MP, 2020; MARD, 2006).
is the long period needed to perform the test and the
occurrence of falsely positive results if the tests are used for
the analysis of individual samples containing higher levels
of naturally occurring antimicrobial agents. Rapid tests
enable to obtain the result of a test in several minutes and
they are highly specific. A majority of the rapid assays were
aimed above all, at the detection of β-lactam antibiotics.
Even in these tests, falsely positive results can occur
(Navrátilová, 2008).
The performance criteria described by the European
Decision 2002/657/EC have to be used for validation of
these tests (Le Breton, Savoy-Perroud and Diserens,
2007).
The quantification of these substances is performed by Figure 1 Reading results in Delvotest® SP-NT.
High-Performance Liquid Chromatography/Tandem Mass
Spectrometry (SVFI, 2021). ECLIPSE 50 combines the principle of agar diffusion tests
with a change in the color of the indicator due to the active
Scientific Hypothesis metabolism of the test microorganism in the absence of an
The number of positive samples decrees during the inhibitor. The Eclipse 50 kit has format microplates. The
observed period. On-farm milk testing with rapid veterinary test sample is dispensed into a well-filled agar medium
drug residue screening tests by farmers or dairies is an containing Bacillus stearothermophillus var. calidolactis.
effective tool to reduce the risk of processing milk Incubation at 65 °C, at which normal growth of the test
contaminated with veterinary drugs. strain, causes the color of the pH indicator to change from
blue to yellow. If they are under investigation in a sample
MATERIAL AND METHODOLOGY of the growth inhibitory substance of the test strain, the
Samples color of the indicator remains blue-violet (MARD, 2009a).
Raw cow milk samples were sampled on the farm by
trained and certified personnel from dairy factories. These Description of the Experiment
persons were trained by the Veterinary and Food Institute in Sample preparation: samples were heated to 40 ±1 °C in
Slovakia in National Reference Laboratory for Milk and the water bath and gently mixed.
Milk Products. The training course “Sampling of raw milk Number of samples analyzed: The total number of
manually and by autosamplers for milk payment as part of analyzed raw cows' milk samples was 452,731.
self-inspection” is accredited by the Ministry of Education, Number of repeated analyses: Each sample was
Science, Research and Sport of The Slovak Republic analyzed once. If a positive result was found, the analysis
(MESRS, 2020). was repeated.
Chemicals Laboratory samples were sampled manually according to
Samples were not preserved or preserved by Azidiol the international standard ISO 707 (ISO, 2008) or by
0.02% (PanReac AppliChem). autosamplers according to the Slovak technical standard
Animals and Biological Material: STN 57 0005 (STN, 2000) with calibrated autosampler.
This research does not involve the use of animals or Samples were stored at 5 °C and sent to one of two Central
biological materials. testing laboratories authorized by the Ministry of
Instruments Agriculture and Rural Development of the Slovak Republic
Laboratories used water-baths 40 °C for sample (MILEX Progres, a.s. Bratislava and EXAMINALA –
preparation and incubators 64 °C ±0.5 °C and 65 ±0.5 °C Dairy Research Institute, Žlina).
for samples incubation. Samples were analyzed by one of the above-mentioned
Laboratory Methods laboratory methods. Results of the analysis were evaluated
The following laboratory method was used: as positive or negative. Overall Results were sent to the
- Delvotest® SP-NT (Supplier: Skar s.r.o. Veterinary and Food Institute Bratislava, National reference
Bratislava, Slovakia); laboratory for milk and milk products in Nitra, Slovakia to
- Eclipse 50 test (Supplier: Merck, Slovakia). be processed and statistically evaluated.
Volume 15 859 2021Potravinarstvo Slovak Journal of Food Sciences
Statistical Analysis Milk control system for the presence of inhibitory
Firstly, the total number of analyzed samples per year was substances
calculated. Secondly, the percentage of positive samples The first level of control
was calculated. These results were plotted on the graph The first stage of control is carried out directly on the
(Figure 2). The reliability equation and the coefficient of R2 farms using rapid screening tests carried out by the farmer
were calculated by Microsoft Excell. or by the driver of the tanker truck before milk is pumped
We have used ONE-WAY ANOVA to evaluate the effect to the tanker and transported to the dairy. However, not all
of the rapid drug residues tests used by farmers. The results farmers or tanker truck drivers check freshly milked milk
were statistically significant at a p-value less than 0.05. directly on the farm. From the dairy's point of view, this
The group of samples from 2001 was considered as a level of control directly at the farmer is interesting because
comparison group of samples because rapid tests were not it eliminates the problem of contamination of milk from
yet widely used in Slovakia in this period. other farmers in the truck, and also it eliminates the transfer
The results of the group of samples from 2020 were of risk and potential problems with the presence of
analyzed against this comparison group of samples from veterinary drug residues to the next stage of the food chain.
2001. Food business operators carry out this control for their own
The statistical evaluation was performed by XLSTAT money to eliminate the risk of supplying milk containing
2021.2 (Addinsoft). antibiotic residues. The purchase of rapid screening tests is
provided directly by farmers or by dairies and distributed to
RESULTS AND DISCUSSION farmers or milk tankers drivers.
The results of the experiment are present in Table 1 and The second level of control
Figure 2. The overall number of analyzed samples per year The second stage of control takes place at the entrance to
decrease continuously from 34,795 (2001) to 12,181 (2020). the dairy as a self-verification check. Following EC
This fact results from a change in legislation due to the Regulation No 852/2004 (EC, 2004a), the established
accession of the Slovak Republic to the European Union, HACCP system and monitoring for the presence of
which took place on 1 May 2004, as well as from the inhibitory substances are performed routinely during milk
subsequent decline in the number of dairy farms and the intake. Each dairy has a customized monitoring system. In
number of dairy cows. In 1989, 559 thousand dairy cows most cases, the laboratory worker will firstly take samples
were bred in Slovakia and milk production reached more from individual farms sampled by the truck driver or
than two billion liters. At present, only less than a quarter of autosampler. Also, it is necessary to take the “tanker
dairy farms are bred in Slovakia, and milk production has sample” which contains milk from all farmers. Usually, this
halved (Pravda 2019). The number of drug residues sample is sampled automatically on farms if the
positive samples significantly (pPotravinarstvo Slovak Journal of Food Sciences
Table 1 Number of samples analyzed in central testing laboratories and number of drug residues positive samples in Slovakia.
Year Number of Number of Number of Percentahe of
analyzed samples negative samples positive samples possitive samples
2001 34 795 34 496 299 0.859
2002 36 050 35 869 181 0.502
2003 33 402 33 210 192 0.575
2004 30 572 30 458 114 0.373
2005 30 324 30 250 74 0.244
2006 27 646 27 547 99 0.358
2007 26 024 25 992 32 0.123
2008 26 297 26 275 22 0.084
2009 23 313 23 286 27 0.116
2010 21 371 21 360 11 0.051
2011 19 475 19 458 17 0.087
2012 18 754 18 746 8 0.043
2013 17 965 17 959 6 0.033
2014 18 918 18 913 5 0.026
2015 17 440 17 436 4 0.023
2016 15 270 15 267 3 0.020
2017 15 321 15 317 4 0.026
2018 14 196 14 191 5 0.035
2019 13 417 13 414 3 0.022
2020 12 181 12 178 3 0.025
Percentage of positive samples of raw cow's milk for the presence of residues of
inhibitory substances in the years 2001 – 2020 in Slovakia
1.000
0.900
0.859
0.800
0.700
0.575
0.600
% of possitive samples
0.502
0.500
0.373
0.400
0.358
0.300
y = -0.0325x + 0.5224
0.244 R² = 0.6725
0.200
0.123
0.116
0.100
0.084 0.087
0.043 0.033 0.250
0.051 0.026 0.023 0.020 0.026 0.0350.220
0.000
2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020
(34 795)(36 050)(33 402)(30 572)(30 324)(27 646)(26 024)(26 297)(23 313) (21371) (19475) (18754) (19965) (18918) (17440) (15270) (15321) (14196) (13417) (12181)
Year and number of samples tested
Figure 1 Percentage of positive samples of raw cow's milk for the presence of residues of inhibitory substances in the years
2001 – 2020 in Slovakia.
Volume 15 861 2021Potravinarstvo Slovak Journal of Food Sciences
The damage must be paid by the farmer who caused such Reference Laboratory for Milk and Milk Products. These
contamination. Usually, the incurred damage is resolved as persons periodically undergo an accredited educational
an insured event. activity every five years and hold a “milk sampler's
In Slovakia or European Union, the legislation does not certificate”, which entitles them to take milk samples. In
specify which laboratory should test samples (EC, 2006). principle, such samples are taken in duplicate. One sample
Even the legislation does not require accreditation of this remains on the farm and the other is used by a dairy
laboratory! In practice, this situation can be handled directly company. The sampling plan created by the dairy for
by the legislation of the country or by the supplier-customer payment must be secret. Farmers must not know the date of
contract. In Slovakia, it is usually tested in one of the two this sampling. Sampler notifies the farmer immediately
accredited central testing laboratories specifically intended when this sample is collected. All samples are sent to a
for milk analysis or in the National Reference Laboratory contracted laboratory (usually a central testing laboratory).
for Milk and Milk Products in Lužianky or by the National If residues of inhibitory substances are detected in the
Reference Laboratory for Antibacterial Substances and second or third stage of control, the dairy shall trace the
Other Pharmacologically Active Substances Dyes and source of the contamination to identify the farm which
Some Prohibited Substances in Dolný Kubín (MARD, caused the contamination.
2020). The farm that caused the contamination will then provide
In practice, dairies usually use the services of accredited financial compensation for milk and costs associated with
laboratories for confirmation of positive samples only the legal disposal of the contaminated milk to all farmers
rarely. They usually immediately consider the sample to be affected by this contamination. From a technical point of
positive. This is how it is solved in most supplier-customer view, single-chamber or multi-chamber tanks can be used
contracts. Confirmation of positive samples can be to transport milk. In most cases, the milk is contaminated in
performed in a contracted accredited central testing the relevant chamber of the entire milk tank into which the
laboratory or one of the above-mentioned national reference milk is pumped is contaminated. In some cases,
laboratories. When the accredited laboratory confirms the contamination of several chambers in the milk tank may
positivity of the sample, then milk is legally disposed of by occur if the capacity of the currently filled chamber is
the authorized company under a decision of the Regional insufficient or by cross-contamination from residues of
Veterinary and Food Administration. contaminated milk left in the hoses and distribution system
Some dairies in Slovakia have recently started sending of the milk tank truck. In the case of using a multi-chamber
milk samples for analysis of milk composition, total tank, it is, therefore, possible to save part of the milk supply,
bacterial count, somatic cells count, and drug residues to since the positive milk is usually only found in one of the
laboratories in Hungary, Austria, Germany, or the Czech chambers of the tank.
Republic. The results from these laboratories are then not In the case of confirmation of a "positive" result for the
statistically processed by National Reference Laboratory for presence of antibiotic residues, the dairy plant shall carry
Milk and Milk Products. Only the results from central out the following:
testing laboratories in Slovakia are processed by NRL. - does not accept (reject) the milk and sends it for
The dairy reports the presence of a positive case of disposal following the rules laid down in Regulation
residues of inhibitory substances to the Regional Veterinary (EC) No. 1069/2009 (EC, 2009b) on animal by-
and Food Administration, which will carry out an inspection products and derived products not intented for human
at the farmer who caused the contamination. consumption. The disposal of Category 3 material is
After the correction, the farmer will request a Regional carried out as follows:
Veterinary and Food Administration for sampling the milk, - applied to land without processing,
which will then be examined in an accredited testing - it can also be disposed of by composting or
laboratory. Only after this analysis can the farmer deliver transformation into biogas.
the milk to the dairy again. - Such disposal is possible only in that case if the
competent authority does not consider it a risk of any
The third level of control disease communicable through that product to humans
The third stage of control is performed in central testing or animals.
laboratories or any other laboratory. Dairies in Slovakia are According to the Decree of the Ministry of Industry and
usually sent milk samples for the inhibitory drug residues Trade of the Slovak Republic No. 148/2012 (MIT SR,
substances test to the contracted one central testing 2012), such disposal must be carried out only under the
laboratory. The frequency of testing is not prescribed in official supervision of a veterinary inspector with his
European legislation (EC, 2006). Usually, two samples per consent. According to EC Regulation no. 142/2011 (EC,
month are collected from each farm. This testing is 2011), the removal of milk by the wastewater stream is
performed as a part of the verification process based on the prohibited.
HACCP plans. Also, the frequency of testing is mentioned Depending on how the buyer has stated in the
in the supplier-purchaser contract. The testing is generally supplier-customer contract with the farmer, he can ensure
carried out in conjunction with the examination of the that another confirmatory test is performed, which identifies
freezing point of the milk because it is recommended to and quantifies the antibiotic and proves whether or not the
analyze not preserved milk samples. On the other hand, for maximum residue limit for this particular antibiotic is
some tests, preserved samples may be used. exceeded. These confirmation tests are usually performed in
Persons taking milk samples for payment purposes, accredited testing laboratory: Veterinary and Food Institute
whether drivers or directly the purchaser's staff, must be Dolný Kubín, National Reference Laboratory for
trained by the Veterinary and Food Institute, National Antibacterial Substances and Other Pharmacologically
Volume 15 862 2021Potravinarstvo Slovak Journal of Food Sciences
Active Substances, Dyes and Certain Prohibited contamination of individual components of the food chain,
Substances. to implement effective measures to eliminate the identified
If the maximum residue limit is exceeded, the milk and deficiencies and prevent their further occurrence. Long-
milk products are disposed of legally. If the maximum term monitoring of the presence of inhibitory substances in
residue limit is not exceeded, milk and products made from milk leads to the finding that almost 95% of cases of
it are considered safe. secondary contamination with inhibitory substances are
According to Kološta (2007), the biggest problem is antibiotic contamination, approximately 2.5% of
mainly residues of antibiotics and sulfonamides, as these are contamination is due to other drugs, especially
mostly used in the treatment of dairy cows. The reference sulfonamides and residual detergent and disinfectant
method for the detection of antibiotics and sulphonamides residues, 1% may cause mycotoxins from feed (nuclear,
in raw milk and heat-treated milk is the CD 91/180 / EEC bulk), 1% may be caused by agrochemical residues or some
method on the principle of microbial inhibition. The preservatives and then only about 0.5% remains on natural
mentioned method was also taken over into the system of inhibitors in milk (Kološta, 2007).
Slovak technical standards STN 57 0531 Determination of Commission Regulation (EC) No. 853/2004 (EC, 2004b)
antibiotics and sulfonamides in raw and heat-treated milk contains specific hygiene rules for food of animal origin.
(STN, 2001). Commission Regulation (EC) No 1662/2006 (EC, 2006)
The journals of the Ministry of Agriculture and Rural defines health requirements for the production of raw milk.
Development of the Slovak Republic state which laboratory Raw milk must come from animals to which no
methods can be used for the official control of the presence unauthorized substances have been administered and the
of inhibitory substances in milk. These are methods based products (medicines or substances) administered have
on the principle of agar diffusion tests with a change in the complied with the withdrawal periods prescribed for those
color of the indicator due to the active metabolism of the products. This Regulation also defines the requirements for
test microorganism (Bacillus stearothermophilus var. milking, transport, and transport hygiene. According to the
Calidolactis) in the absence of an inhibitor. The Regulation, it is necessary to identify animals that have
determination of residues of inhibitory substances in milk undergone treatment which could cause the excretion of
and milk products can therefore be carried out in central drug residues in milk and colostrum, and that milk and
testing laboratories by the following methods approved by colostrum obtained from such animals should not be used
the Ministry of Agriculture and Rural Development of the for human consumption before the expiry of the prescribed
Slovak Republic: withdrawal period. Dips or sprays pods will be used only
- Delvotest® SP-NT (MARD SR, 2006), after authorization or registration. Based on these
- Eclipse 50 (MARD SR, 2009a) or requirements, primary milk producers must put in place
- Kalidos TB MP (MARD SR, 2009b). systems to control the treatment of dairy cows.
Also, there is a In the past, the State Veterinary and Food Administration
- CMT+ test (Climax, 2021), which has been published a guideline for control of raw milk in farms for
verified by the National Reference Laboratory for the production of raw milk (SVFA, 2009), today the control
Milk and Milk products, but it is not listed in the is based on the requirements of the current European
list of approved methods of the Ministry of legislation (EC, 2017; EC, 2019), Title IV, Article 49
Agriculture and Rural Development of the Slovak Control of milk and colostrum production holdings and
Republic. Article 50 Control of milk and colostrum.
When testing raw cow's milk for payment, central testing The Regional Veterinary and Food Administration checks
laboratories in the Slovak Republic (MILEX Progres, as the food business operator whether, according to EC
Bratislava and EXAMINALA – Dairy Research Institute in Regulation no. 1662/2006 (EC, 2006) has procedures in
Žilina) use a diffusion test to determine residues of place to ensure that raw milk is not placed on the market if
inhibitory substances, which lasts 3 hours: it contains antibiotic residues in quantities which, for any of
Delvotest® SP-NT. This test is used also by the National the substances listed in Annexes I and III to Regulation
Reference Laboratory for Milk and Milk Products in Nitra (EEC) No. 2377/90 (EC, 1990) exceeds the value permitted
(NRLM) (Zajác and Čapla, 2010). under that Regulation or if the combined total antibiotic
However, it is not possible to determine the exact amount residue level exceeds any maximum permitted value.
of antibiotic residues or a specific species using these During the official control, the inspector fills in the
microbiological or antigen methods. prescribed official control record, in which he states the
Veterinary and Food Institute Dolny Kubin, National number of reports of positive cases of residues of inhibitory
Reference Laboratory for Antibacterial Agents and Other substances in raw milk. It also checks whether the supplier
Pharmacologically Active Substances, Dyes and Certain and the relevant regional veterinary and food administration
Prohibited Substances quantify veterinary drug residues by of the Slovak Republic have been informed about
HPLC/MC method (SVFI, 2021). unsatisfactory results of raw milk. In case of unsatisfactory
results of raw milk, the inspector checks the documentation,
The fourth degree of control the date of detection (notification from the Central testing
The State Veterinary and Food Administration of the laboratory, the State Veterinary, and Food Institute, the own
Slovak Republic annually compiles the National Plan for laboratory, or any laboratory), the date of written
the Control of Residues in Live Animals and Products of notification to the relevant Regional Veterinary and Food
Animal Origin following the applicable legislation of the Administration and the raw milk supplier.
EU (EC, 2017; EC, 2019). The aim of the control through The food business operator must therefore take measures
this plan is to obtain an overview of the current state of to remedy this situation and should not place milk
Volume 15 863 2021Potravinarstvo Slovak Journal of Food Sciences
containing residues of inhibitory substances on the market. Table 2 Prohibited substances detected in milk in EU.
In the case of residues of inhibitory substances, the Year Drug residue Number of Countries Source
legislation does not further specify what measures the food non- reporting
compliant non-
business operator has to take. The legislation does not results compliant
specify how the control body should proceed, nor does it results
specify whether samples are to be taken to prove 2019 Chloramphenicol 3 Italy, (EFSA,
remediation and where (what laboratory) these are to be Latvia, 2021)
Poland
tested. However, according to the methodological guideline 2018 Chloramphenicol 2 Poland, (EFSA,
(SVFA SR, 2009), inspectors of Regional Veterinary and Spain 2020)
Food Administrations are instructed as follows: the 2017 Chloramphenicol 1 Croatia (EFSA,
producer of raw milk must start procedures to ensure that 2019)
raw milk is not placed on the market, i.e. unless corrective 2017 AHD (1-aminohydantoin) 1 Croatia (EFSA,
2019)
measures have been taken and it should be proved that raw 2017 AMOZ (5- 1 Croatia (EFSA,
milk does not exceed the maximum permitted level of the methylmorpholino-3- 2019)
combined total antibiotic residues. Food business operators amino-2-oxazolidone)
2017 AOZ (3-amino-2- 1 Croatia (EFSA,
must not place such milk on the market. In practice, this oxazolidone) 2019)
means that after the situation has been remedied, the farmer 2017 SEM (semicarbazide) 1 Croatia (EFSA,
must have a sample of milk examined at his own expense in 2019)
the laboratory (it is not defined what kind of laboratory, but 2016 Chloramphenicol 3 Poland (EFSA,
usually it is agreed with the inspector what laboratory 2018)
2016 Hydroxymetronidazole 1 Germany (EFSA,
should be used for this testing. Almost it is an accredited (MNZOH) 2018)
central testing laboratory or any accredited laboratory of the 2015 Chloramphenicol 2 Latvia, (EFSA,
Veterinary and Food Institute – NRL for milk and milk Poland 2017)
2014 Chloramphenicol 2 Latvia, (EFSA,
products or NRL for Antibacterial Substances). If the result
Poland 2016)
of the test shows that the milk does not contain antibiotic 2013 Chloramphenicol 3 Latvia, (EFSA,
residues, it may place it on the market. Slovenia, 2015)
Control of antibiotic residues is necessary to protect the Slovakia
2012 Chloramphenicol 1 Spain (EFSA,
reputation of milk as safe food and to prevent exposure of 2014)
the consumers to drug residues. Governmental authorities 2011 Chloramphenicol 1 Spain (EFSA,
which are obliged to organize supervision and control 2013)
systems by state supervision agencies must provide these 2010 Chloramphenicol 3 Czech (EFSA,
requirements (Popelka, et al., 2003). Republic, 2012)
Spain
EU countries must monitor food of animal origin for the 2009 Chloramphenicol 1 Slovakia
presence of residues. The legislation governing the design Note: the list of the reports is present on the European
and implementation of residue monitoring plans is as Commission website (EC, 2021).
follows (EC, 2021):
- Directive 96/23/EC - Annexes are in force and establish The specific species of residues detected are listed in
the sampling frequency and range of substances to be tested Table 3. There are several specific groups of residues
for. according to the EU legislation: A (1-6), B (1-3). European
- Decision 97/747/EC - Annexes are in force and establish Union member states report all positive samples from the
additional sampling frequencies for milk. monitoring of veterinary medicinal product residues and
- Regulation (EU) 2021/808 - Rules for the validation of other substances in live animals and animal products
analytical methods used in the residue monitoring plan and including milk (EFSA, 2021).
official sample treatment (Laboratory Analysis). The Slovak Republic has performed the monitoring and
Specific EU legislation outlines the laboratory analysis the targeted sampling according to the EU legislation. The
and correct interpretation of results: number of positive samples was:
- Regulation (EU) 2021/808 establishes Criteria and - 2013: Chloramphenicol (1 positive sample)
procedures to validate analytical methods, ensuring the (EFSA, 2015),
quality and comparability of the results of official - 2012: Benzylpenicillin (Penicillin G) (1 positive
laboratories. Common criteria for the interpretation of test sample) (EFSA, 2014),
results. Minimum required performance limits (MRPL) for - 2009: Chloramphenicol (1 positive sample)
analytical methods to detect substances without a maximum (EFSA, 2011).
limit. This is relevant for substances that are not authorized Positive samples present in Table 1 are not included in
or that are specifically prohibited in the EU. Also sampling these reports (Table 2), because they were detected in the
procedures and official sample treatment. self-control by dairies and were not sampled as a part of the
Commission Decision 97/747/EC lays down that the targeted monitoring performed by the Veterinary and Food
annual number of samples taken should be one per 15,000 Administration of the Slovak Republic.
tonnes of annual milk production, with a minimum of 300 The monitoring system in Slovakia is functional and will
samples (EFSA, 2019). detect possible contamination of milk with residues of
The overview of the non-compliant results for prohibited veterinary drugs. Dairies in Slovakia process only milk that
substances is present in Table 2. Chloramphenicol is the does not contain residues of veterinary drugs.
most dominant prohibited substance detected in milk in
European Union.
Volume 15 864 2021Potravinarstvo Slovak Journal of Food Sciences
Table 3 Overview of the inhibitory substances detected in milk in EU 2015 – 2019.
Year Overview of the substances detected Source
2019 In group A, there were three non-compliant samples and non-compliant results reported in group (EFSA,
A6 (chloramphenicol), by three countries. For antibacterials (B1), seven countries reported a total 2021)
of 11 non-compliant samples and results. In the group B2, there were 5 non-compliant samples and
results for anthelmintics (B2a) and 19 noncompliant samples and results for NSAIDs (B2e),
reported by three and eight countries, respectively. In the group B3, there were three non-compliant
samples and results for mycotoxins (B3d), relating to Aflatoxin M1, reported by two countries. For
organochlorine compounds, including PCBs (B3a) there was one non-compliant sample (2 non-
compliant results) reported by one country. More information on the specific substances identified
and the number of non-compliant results reported by each country is given in Appendix A. Detected
substances: Chloramphenicol, Aminosidin (Paromycin, Paromomycin), Amoxycillin, Ampicillin,
Benzylpenicillin (Penicillin G), Dihydrostreptomycin, Doxycycline, Rifaximin, Closantel,
Ivermectin, Moxidectin, Oxyclozanide, Acetaminophen (Paracetamol), Antipyrin-4-Hydroxy,
Diclofen (Diclofenac), Salicylic acid, TEQ Dioxin-like PCBs LB, Aflatoxin M1.
2018 In the group A, there were two non-compliant samples (two non-compliant results) for group A6 (EFSA,
(chloramphenicol), reported by two Member States. For antibacterials (B1), four Member States 2020)
reported a total of four non-compliant samples (and results). In the group B2, there were 18 non-
compliant samples and results: 6 for anthelmintics (B2a) and 12 for NSAIDs (B2e). In the group
B3, there was three non-compliant samples and results for chemical elements (B3c), relating to
lead, reported by two Member States. More information on the specific substances identified and
the number of non-compliant results reported by each Member State is given in Appendix A.
Detected substances: Chloramphenicol, Aminosidin (Paromycin, Paromomycin), Amoxycillin,
Cloxacillin, Oxytetracycline, Closantel, Ivermectin, Albendazole, Sulphoxide, Albendazole
sulphone, Albendazole 2-amino sulphone, Diclofen (Diclofenac), Salicylic acid, Lead (Pb).
2017 In the group A, there were two non-compliant samples (five non-compliant results) in group A6 (EFSA,
reported by one Member State (one result each for AHD, AMOZ, AOZ, chloramphenicol and 2019)
SEM). For antibacterials (B1), seven Member States reported a total of 19 non-compliant samples
(20 noncompliant results). In the group B2, there were 54 non-compliant samples and results: 11
for anthelmintics (B2a) and 43 for NSAIDs (B2e). In the group B3, there were 11 non-compliant
samples and results for mycotoxins (B3d) (all aflatoxin M1). More information on the specific
substances identified and the number of non-compliant results reported by each Member State is
given in Appendix A. Detected substances: AHD (1-aminohydantoin), AMOZ (5-
methylmorpholino-3-amino-2-oxazolidone), AOZ (3-amino-2-oxazolidone), Chloramphenicol,
SEM (semicarbazide), Ampicillin, Benzylpenicillin (Penicillin G), Cefalonium, Ciprofloxacin,
Cloxacillin, Doxycycline, Florfenico, Oxytetracycline and its 4-epimer, tetracycline and its 4-
epimer, Trimethoprim, Tulathromycin, Clorsulon, Closantel, Ivermectin, Levamisole, Nitroxinil,
Ketotriclabendazole, Oxfendazole sulphone, Triclabendazolsulfon, Diclofen (Diclofenac),
Salicylic acid, Aflatoxin M1.
2016 Detected substances: Chloramphenicol, Hydroxymetronidazol (MNZOH), Amoxycillin, (EFSA,
Benzylpenicillin (Penicillin G), Danofloxacin, Gentamicin, Tetracycline, Tilmicosin, Ivermectin, 2018)
Triclabendazole, Diclofen (Diclofenac), Salicylic acid, 2,4-Dichlorophenoxybutyric acid, Lead,
Aflatoxin M1.
2015 Detected substances: Chloramphenicol, Amoxycillin, Benzylpenicillin (Penicillin G), Cefalexin (EFSA,
(Cefalexin Anhydrate), Cefquinom, Cloxacillin, Enrofloxacin, Kanamycin, Spiramycin, 2017)
Tetracycline, Albendazol, Closantel, Ivermectin, Diclofen (Diclofenac), Lead Pb, Aflatoxin M1.
CONCLUSION samples from all milk deliveries in central testing
The system of monitoring of inhibitory drug residues in laboratories, the responsibility for control and testing
raw cow's milk applied in Slovakia is effective, it can detect remains on the dairies. Every supply of raw cow's milk is
the presence of these substances before milk is processed in tested for the presence of residues of inhibitory substances
dairies. The total incidence of these substances in 2020 was in each dairy company in Slovakia. The role of the State
0.025% of all 12,181 samples tested. A total number of Veterinary and Food Administration of the Slovak Republic
452,731 samples have been tested over the last twenty years is to monitor and control whether food business operators
in central testing laboratories. The number of drug residues implement measures to prevent the placing on the market of
positive samples significantly (pPotravinarstvo Slovak Journal of Food Sciences
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p. 3041-3053. https://doi.org/10.3168/jds.S0022- We would like to thank you to the Veterinary and Food
0302(93)77644-4
Institute, Veterinary and Food Institute Bratislava, Central
VFA SR, 2009. Metodický pokyn č. 14/2009 Štátnej
testing laboratory Examinala in Žilina and Central testing
veterinárnej a potravinovej správy slovenskej republiky.
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Veterinárne kontroly surového mlieka v chovoch na produkciu
surového mlieka. (Methodical instruction no. 14/2009 of the
State Veterinary and Food Administration of the Slovak Conflict of Interest:
Republic. Veterinary checks on raw milk in raw milk The authors declare no conflict of interest.
production holdings). (In Slovak).
ZAJÁC, P., ČAPLA, J. 2010. Prehľad výskytu rezíduí Ethical Statement:
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republike za roky 2001 až 2009 (Overview of the occurrence an ethical statement.
of residues of inhibitory substances in raw cow's milk in the
Slovak Republic for the years 2001 to 2009). In: Bezpečnosť a Contact Address:
kontrola potravín : zborník prác z medzinárodnej vedeckej *Peter Zajác, Slovak University of Agriculture in Nitra,
konferencie (In: Food Safety and Control: Proceedings of the Faculty of Biotechnology and Food Sciences, Institute of
International Scientific Conference), Nitra, Slovakia. 24-25 Food Sciences, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia,
March, 2010 : Slovak University of Agriculture in Nitra, p. Tel.: +421376414371,
199-200, ISBN 978-80-552-0350-8. (In Slovak) E-mail: peter.zajac@uniag.sk
ZAJÁC, P., GOLIAN, J., ČAPLA, J. 2007a. Rezíduá ORCID: https://orcid.org/0000-0002-4425-4374
inhibičných látok v surovom kravskom mlieku v Slovenskej Stanislava Zubrická, State Veterinary and Food Institute,
republike v roku 2007. (Residues of drugs in raw cow milk in Veterinary and Food Institute Bratislava, Botanická ulica č.
Slovak Republic in year 2007). In: Bezpečnosť a kontrola 15, 842 52 Bratislava, Slovakia, Tel.: +421905393419,
potravín : zborník prác z medzinárodnej vedeckej konferencie
E-mail: nrlm@svuba.sk
(Food safety and control: proceedings of an international
ORCID: https://orcid.org/0000-0003-0387-8575
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ZAJÁC, P., GOLIAN, J., VÁCZIOVÁ, Z. 2006. Výsledky Food Sciences, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia,
vyšetrenia surového kravského mlieka na RIL v SR za roky Tel.: +421376414371,
2001 – 2005. (Results of examination of raw cow 's milk at RIL E-mail: jozef.capla@uniag.sk
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Bezpečnosť a kontrola potravín II. Diel: zborník prác Jozef Čurlej, Slovak University of Agriculture in Nitra,
z medzinárodnej vedeckej konferencie. (In: Food Safety and Faculty of Biotechnology and Food Sciences, Institute of
Control II. Part: proceedings of an international scientific Food Sciences, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia,
conference), Nitra, Slovakia : Slovak Agriculture and Food Tel.: +421376415825,
Inustry Chamber, 5. – 6. April 2006, p. 299-305, ISBN 80- E-mail: jozef.curlej@uniag.sk
8069-682-9. (In Slovak) ORCID: https://orcid.org/0000-0003-0039-5332
ZAJÁC, P., GOLIAN., J., VÁCZIOVÁ, Z. 2007b. Rezíduá Maroš Drončovský, Slovak Dairy Research Institue, Dlhá
inhibičných látok v surovom kravskom mlieku v roku 2006 95, 010 01 Žilina, Slovakia, Tel.: +421417072113,
a trendy ich výskytu. (Residues of inhibitory substances in raw E-mail: droncovsky@vumza.sk
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